Summary

MELK is a serine/threonine kinase involved in several cell processes, including the cell cycle, proliferation, apoptosis and mRNA processing. However, its function remains elusive. Here, we explored its role in the Xenopus early embryo and show by knockdown that xMELK (Xenopus MELK) is necessary for completion of cell division. Consistent with a role in cell division, endogenous xMELK accumulates at the equatorial cortex of anaphase blastomeres. Its relocalization is highly dynamic and correlates with a conformational rearrangement in xMELK. Overexpression of xMELK leads to failure of cytokinesis and impairs accumulation at the division furrow of activated RhoA – a pivotal regulator of cytokinesis. Furthermore, endogenous xMELK associates and colocalizes with the cytokinesis organizer anillin. Unexpectedly, our study reveals a transition in the mode of cytokinesis correlated to cell size and that implicates xMELK. Collectively, our findings disclose the importance of xMELK in cytokinesis during early development and show that the mechanism of cytokinesis changes during Xenopus early development.

In human cells and Xenopus embryos, MELK catalytic activity is correlated with its phosphorylation and is maximal during mitosis (Blot et al., 2002; Davezac et al., 2002). We have shown that, in M phase, MELK is phosphorylated at multiple sites (Badouel et al., 2006) and that the two major mitotic kinases, MPF (cyclin-B–CDK1 complex) and the mitogen-activated protein kinase (MAPK) ERK2, participate in these phosphorylations and enhance MELK activity in vitro. MELK is potentially involved in cell cycle progression through association with CDC25 (Davezac et al., 2002; Mirey et al., 2005). This protein phosphatase controls entry into mitosis by dephosphorylating and activating the CDK1–cyclin-B complex. The subcellular localization of MELK is also cell cycle dependent as a fraction of MELK protein is redistributed at the cell cortex specifically during anaphase and telophase (Chartrain et al., 2006). Together, these results indicate that mitosis is crucial in MELK regulation; however, the function of Xenopus MELK (xMELK) remains elusive.

In this study, we asked how MELK is involved in mitosis. We investigated the role of xMELK in early embryos in which very rapid cell cycles comprising only S- and M-phases occur in the absence of gene transcription until the mid-blastula transition (MBT) (Newport and Kirschner, 1982a; Newport and Kirschner, 1982b). xMELK is encoded by a maternal mRNA, and its expression, phosphorylation and catalytic activity are tightly controlled during oocyte maturation and early embryonic cleavage (Badouel et al., 2006; Blot et al., 2002; Paris and Philippe, 1990). These fine controls suggested that xMELK might have an important role in early embryos.

Our results reported here show that perturbing xMELK expression by either knockdown or overexpression leads to abortive cell division in embryos. Shortly before cells start cytokinesis, endogenous xMELK as well as other essential cytokinetic proteins accumulate in a narrow band at the equatorial cortex. A FRET-based probe shows that a conformational change correlating with xMELK activation accompanies relocalization of the kinase at the cell cortex. Overexpression of active xMELK, in contrast to a kinase-dead mutant, leads to defective constriction of the cytokinetic ring. xMELK overexpression induces a marked decrease in accumulation of activated Rho GTPase at the cleavage furrow, thus providing a direct explanation for the xMELK-induced failure of cytokinesis. Finally, we show that xMELK is associated with anillin, a crucial cytokinetic protein, and that the two proteins colocalize at the equatorial cortex. Interestingly, our study also reveals that a developmentally regulated transition involving xMELK occurs in the mode of cytokinesis during early embryogenesis.

Results

During Xenopus oogenesis and early embryogenesis, xMELK expression, phosphorylation and activity are tightly controlled (Badouel et al., 2006; Blot et al., 2002; Paris and Philippe, 1990). Indeed, during oocyte maturation, xMELK protein levels increase by approximately 2.5 fold, and xMELK is phosphorylated, inducing a decrease in its electrophoretic mobility (Fig. 1A). After
fertilization, xMELK is rapidly dephosphorylated (Blot et al., 2002), and, during the first cell cycle, its levels are transiently decreased by approximately 50%. In cleaving embryos, the levels of xMELK are similar to those present in eggs and remain unchanged during early development. Such sophisticated expression controls suggested that xMELK could have a function during the cleavage period that follows fertilization. Therefore, to determine this function, we altered xMELK expression in early development. As Xenopus eggs contain a supply of maternal xMELK protein accumulated during oocyte maturation (Fig. 1A), we performed xMELK knockdown before fertilization. This relies on the inhibition of xMELK neosynthesis during oocyte maturation by inducing specific mRNA degradation with two antisense phosphorotioate-modified deoxyoligonucleotides (AS9 and AS11 xMELK). The host transfer method (Heasman et al., 1991) was used to obtain embryos from in-vitro-cultured full-grown oocytes injected with AS deoxyoligonucleotides. These interfered neither with the oocyte maturation (Table 1 and supplementary material Fig. S1) nor with the extrusion of the first (data not shown) and the second polar body after release of meiosis II by parthenogenic activation (supplementary material Fig. S1). When compared with uninjected oocytes and oocytes treated with a control AS (‘AS Co’), only AS deoxyoligonucleotides against xMELK induced xMELK mRNA degradation in oocytes (Fig. 1B), leading to a marked decrease of xMELK protein levels in embryos (Fig. 1C). Time-lapse video-recording showed that the large majority of embryos injected with AS Co cleaved as expected (Fig. 1D and Table 1). By contrast, AS9- and AS11-xMELK-treated embryos either did not cleave or, for the majority, presented abortive cleavages (Fig. 1D and Table 1). Abortive cleavage furrows of AS-xMELK-injected embryos appeared at the same time as in controls. Moreover, surface contraction waves, a marker of cell-cycle progression (Hara et al., 1980), occurred with the same frequency as controls, indicating that the cell cycle progression is not impaired in AS-xMELK embryos. Altogether, these results demonstrate that xMELK is a maternal-effect gene required for early embryonic cleavages and that xMELK knockdown impairs the progression, but not the initiation, of the cleavage furrow.

Abortive cell divisions induced by xMELK knockdown. (A) xMELK expression was followed by western blot with anti-xMELK antibodies during in vitro oocyte maturation released by progesterone [Pg, prophase arrested (PI) and metaphase arrested (MII) oocytes] and at different times post-fertilization (Pf.). xMELK to β-tubulin (used as a loading control) ratios are indicated. In MII oocytes, xMELK is phosphorylated and, consequently, its electrophoretic mobility is decreased compared with that of PI and embryos (Blot et al., 2002; Badouel et al., 2006). (B) Northern blot analysis with an xMELK probe of oocytes treated with 2 or 4 ng of AS Co, AS9, AS11 oligos or untreated (−). Eg7 was used as a loading control. (C) Western blot analysis with anti-xMELK antibodies of individual AS Co (a and b), AS11 (c, d and e) and AS9 (f and g) MELK-treated embryos. xMELK to actin (used as a loading control) ratios are indicated. (D) Time-lapse images of AS Co, AS9 and AS 11 embryos. Arrowheads point to the furrows of the first and second abortive divisions. Time is in minutes.

Cytokinesis progression is inhibited by xMELK overexpression

To gain insight into the xMELK function, we also overexpressed xMELK and examined the effects of increased levels in early embryos. In-vitro-transcribed mRNAs coding xMELK were microinjected into a single blastomere of two-cell-stage embryos. xMELK overexpression efficiently induced inhibition of blastomere division, leading to the appearance of abnormally large cells in all embryos (Fig. 2A). The expressivity of the xMELK-induced phenotype was clearly dose dependent. Accordingly, accumulation of xMELK protein increased with the amount of injected xMELK mRNA (Fig. 2A). Inhibition of cell division was not observed when mRNAs encoding enhanced green fluorescent protein (EGFP; Fig. 2A) or the two Xenopus xMELK-related protein kinases xPAR-1A and xPAR-1BX were used (Ossipova et al., 2005) (data not shown). Moreover, expression of an xMELK mutant (xMELK K/R), the activity of which is largely reduced compared with that of the wild-type xMELK (Blot et al., 2002) did not affect cell division (Fig. 2A). This indicates that, upon xMELK overexpression, the kinase activity is necessary for inhibition of cell division. In numerous instances, it has been reported that expression of a dead kinase has a dominant-negative effect. This
is generally due to the titration by the mutant of an activating partner, leading to inactivation of the endogenous kinase, or to the sequestration of its substrate. Therefore, the absence of a dominant-negative effect of xMELK K/R suggests that xMELK does not stably associate with an activator or a substrate to perform its role in cell division or that the point mutation (K/R) directly or indirectly destabilizes a putative interaction.

Overexpression of xMELK induces abortive cell divisions. (A) Embryos microinjected with 5 ng or 1 ng of xMELK, 1 ng of xMELK K/R or 5 ng of EGFP mRNAs. Blastulas are shown on the left and gastrulas on the right (only the gastrula stage is shown for xMELK K/R). Dotted lines indicate large undivided cells. xMELK and xMELK K/R constructs are indicated: N, catalytic; M, median; and C, C-terminal domains. Western blots show the levels of expressed proteins. (B) Uninjected (Co) and xMELK-overexpressing (xMELK) embryos were fixed and stained for DNA (blue), F-actin (red) and lamin or β-tubulin (green), as indicated. Scale bars: 10 μm. (C) Still frames of time-lapse confocal microscopy showing embryos coinjected with xMELK and the GFP–ABD fluorescent probe mRNAs. Projections of six confocal 1-μm sections are shown. Arrowheads indicate division furrows that ultimately regress (asterisks). Time is indicated in minutes. Scale bar: 50 μm.

Analyses by confocal microscopy revealed that large undivided cells contain multiple nuclei and supernumerary centrosomes, indicating that these cells present a cytokinesis defect, whereas the cell cycle is not impaired (Fig. 2B). To define more precisely the induced defect, embryos overexpressing xMELK were followed by time-lapse confocal microscopy. To visualize the periphery of the cells, GFP–ABD, a fluorescent protein having affinity for actin (Lenart et al., 2005), was coexpressed with xMELK. In these embryos, the cleavage furrows started to ingress but failed to complete cell division and finally regressed (Fig. 2C). This shows that xMELK overexpression, as well as xMELK knockdown, inhibits the progression, rather than the initiation, of cytokinesis.

Collectively, the results of knockdown and overexpression experiments demonstrate that xMELK is important for the progression of cytokinesis and that its levels must be finely tuned for successful cytokinesis.

Endogenous xMELK is localized at the cell cortex and at the division furrow

As alterations in the levels of xMELK induce cytokinesis failure, we examined endogenous xMELK localization during cell division in Xenopus laevis embryos with a previously characterized affinity-purified antibody against xMELK (Blot et al., 2002; Chartrain et al., 2006). During the first and the second embryonic divisions, xMELK was detected at the cell cortex and at the division site (Fig. 3A). As expected, the small GTPase RhoA was also detected at the cleavage furrow. Interestingly, xMELK colocalizes with RhoA (Fig. 3A, merge panels), indicating that its accumulation at the division site is an early event during cytokinesis. A second antibody against xMELK gave the same result (data not shown). To confirm that the signal detected with antibodies is specific to xMELK, we performed immunofluorescence on embryos treated with either AS Co or AS11 xMELK and fixed when the first division furrow started to ingress. In AS11 xMELK treated embryos, the signal detected with the antibody to xMELK at the cell cortex and the division furrow was largely reduced compared with that of AS Co embryos. In these conditions, even if the furrow was not formed perfectly in AS11 embryos, RhoA was still localized at the division site (Fig. 3B). This demonstrates that the signal detected with the antibody to xMELK is specific and that its accumulation at the furrow is not necessary for RhoA accumulation.

Later in development, at blastula stage 7 (Nieuwkoop and Faber, 1956), xMELK was localized at the lateral cell cortex independently of the cell cycle phase (Fig. 3C, c2). xMELK was also concentrated in a narrow band at the surface of blastomeres during anaphase (Fig. 3C, c1). The xMELK band was already detectable at the equatorial cortex before ingression of the furrow started (Fig. 3D, left), demonstrating that xMELK is an early marker of the cleavage site. As the furrow ingresses, xMELK stays associated with the furrow and moves inwards (Fig. 3D, middle and right). In post-MBT
embryos at the blastula stage 9 and the gastrula stage 11, xMELK was still localized at the cell cortex, but the xMELK equatorial band was no longer detectable (Fig. 3C, c5). Instead, a slightly more intense xMELK signal was sometimes observed at the site of cell constriction in the late blastula (stage 9, Fig. 3C, arrow in c4). This is not specific only to xMELK as F-actin and the myosin heavy chain (MHC) have an identical behaviour (supplementary material Fig. S2A and S2B), demonstrating that a change in the mode of cytokinesis progression occurs during early development concomitantly with the MBT. We also observed that an inversion in the ingression direction of furrows occurs between stage 9 and stage 11. Indeed, at blastula stage 9, whereas the apex of blastomeres has already ingressed, more deeply in cells, the basolateral membrane only shows the beginning of a constriction (Fig. 3C, compare c3 and c4). Conversely, at the gastrula stage 11, the basolateral membrane has already ingressed, whereas their surface is not yet cleaved (Fig. 3C, compare c5 with c6 and see the corresponding orthogonal view and supplementary material Movie 1). This was observed for all proteins analyzed (supplementary material Fig. S2B), demonstrating that this is not an artefact due to xMELK antibodies. At the MBT, many maternal proteins are replaced by zygotically expressed ones, and this is frequently accompanied by a change in their expression levels. Therefore, a decrease in F-actin, MHC and xMELK levels in stage 11 embryos could explain why these proteins are no more concentrated at the equatorial cortex. So, we analysed by western blotting the levels of these proteins in embryos at stages 7, 9 and 11. The results show that the xMELK level in gastrula embryos still represents 51% of the level in blastula (supplementary material Fig. S3A). By contrast, the level of F-actin did not change, and the level of MHC even slightly increased. Accordingly, in living gastrula embryos, EGFP–xMELK was not concentrated at the equatorial cortex (see above and supplementary material Fig. S3B). This demonstrates that, in gastrula embryos, the absence of F-actin, MHC and xMELK concentration at the division site is not directly correlated to their expression levels and indicates that a currently unknown mechanism(s) controls their localization during development. Altogether, these results show that xMELK localization at the equatorial cortex is precisely controlled during development and that the equatorial band is specific to large cells of pre-MBT embryos. The precise localization of xMELK at the division furrow, in addition to the division defect induced by xMELK knockdown and overexpression, strongly suggested a role for xMELK in cytokinesis in cleaving embryos.

Endogenous xMELK is localised at the cell cortex and division furrow. (A) Albino embryos were fixed when the first (top raw) and second (bottom raw) division furrows started to ingress. Indirect immunofluorescence was performed with antibodies against xMELK (green) and RhoA (red). Projections of 22 confocal 0.5-μm sections are shown. Pictures were merged to visualize colocalization of xMELK with RhoA at the division furrow (merge). The dotted line indicates the first division. Scale bar: 100 μm. (B) AS-Co- and AS11-treated embryos were fixed when the first division furrow started to ingress. Indirect immunofluorescence was processed as in (A). For each condition, projections of 26 confocal 0.5-μm sections are shown. Scale bar: 100 μm. (C) Blastula stage 7 (c1 and c2), blastula stage 9 (c3 and c4) and gastrula stage 11 (c5 and c6) albino embryos were fixed, processed for indirect immunofluorescence with antibody against xMELK (green) and stained for DNA (blue). Surface (Z0, c1, c3 and c5) and deeper (distance in μm relative to Z0; c2, c4 and c6) single optical sections are shown. Asterisks indicate dividing cells in stage-9 and -11 embryos. At the right is shown an orthogonal projection of a dividing cell (c5–c6). The plan of orthogonal projection is symbolized by white lines on the two confocal planes shown in panel c5 and c6, and reversely white dotted lines on the orthogonal projection indicate the position of the two confocal planes shown in panels c5 and c6. (D) Single optical sections of the surface of three distinct blastomeres (blastula stage 7) at several stages of cytokinesis fixed as in C. White lines represent the plan of orthogonal projections shown under pictures. Black and open arrowheads indicate, respectively, cell division sites and lateral cortex. Scale bar: 50 μm.

xMELK localization at the cell cortex and at the division furrow is highly dynamic

To determine the dynamics of xMELK localization, EGFP–xMELK fusion proteins were expressed in embryos and followed by time-lapse confocal microscopy. To examine xMELK dynamics under conditions where cell division proceeds normally, we first used the EGFP–XMELK K/R mutant that does not perturb cytokinesis.
Before cells started to divide, fluorescence emitted by the EGFP–XMELK K/R was diffuse, resembling that of EGFP-expressing embryos (compare Fig. 4A with Fig. 4C). Just before the cells started dividing, a thin fluorescent line corresponding to the cell cortex appeared in addition to a faint equatorial band (arrowheads in Fig. 4A and supplementary material Movie 2). While cells progress through mitosis, the fluorescence intensity of this EGFP–XMELK K/R band increased. The EGFP–XMELK K/R band corresponds exactly to the division site and moves inward with ingressing furrows. These results are in agreement with endogenous xMELK immunolocalization in fixed embryos. When cells completed division, the fluorescence came back to the initial state (Fig. 4A). Thus, the dynamic study in whole embryos shows that xMELK is already detectable at the presumptive division site approximately two minutes before membrane ingression. Wild-type EGFP–XMELK presented an identical dynamic localization at the cleavage furrow and at the cell cortex; however, the EGFP–XMELK band became unfocused and, correlating with this, failures of cytokinesis were observed (Fig. 4B and supplementary material Movie 3). The fast relocalization of EGFP–XMELK at the cell cortex was also observed in gastrula embryos. In agreement with results obtained by immunofluorescence on fixed embryos (Fig. 3C), the basolateral membrane was already divided, whereas the surface was not cleaved and EGFP–XMELK was not accumulated at the cell division site (supplementary material Fig. S3C). Together, these results further demonstrate that, in early embryos, xMELK marks the division furrow position before ingression and that its relocalization at the cortex and at the division site is a highly dynamic event.

Dynamics of xMELK localization. (A,B) Still frames of time-lapse confocal microscopy showing, respectively, blastomeres expressing EGFP–XMELK K/R (A) and EGFP–XMELK (B). Projections of 10 confocal 2-μm sections are shown. Arrowheads point to the xMELK band that coincides with the cleavage furrow. Arrows point to unfocused xMELK. Asterisks indicate cells that failed to divide. Time is indicated in minutes. Scale bar: 50 μm. (C) Blastomeres expressing EGFP alone. Arrowheads point to cell division sites. Time is in minutes. Scale bar: 50 μm.

xMELK localization correlates with its conformational rearrangement

It was previously shown that MELK is auto-inhibited by its C-terminal domain and that deletion of this domain results in an increased catalytic activity in vitro (Beullens et al., 2005) (data not shown). The widespread mechanism of kinase auto-inhibition by intramolecular folding was proposed to apply for MELK (Beullens et al., 2005) and Kin2, a MELK-related kinase in yeast (Elbert et al., 2005). Therefore, we asked whether conformational changes in xMELK occur during cell division in Xenopus embryos and we explored the possibility of a correlation between xMELK relocalization at the cleavage furrow and a conformational rearrangement by using intramolecular fluorescence resonance energy transfer (FRET). A FRET-based probe corresponding to xMELK K/R sandwiched between the cyan fluorescent protein (CFP) donor and the yellow fluorescent protein (YFP) acceptor was constructed (Fig. 5A). The FRET probe was expressed in embryos and followed by time-lapse confocal microscopy. The highest YFP:CFP emission ratio, reflecting a closed configuration, was observed when the protein was localized in the cytoplasm and it did not change across the cell cycle (Fig. 5B). A smaller YFP:CFP ratio was observed for the protein localized at the cell cortex, either at the cell surface outside of the division site or located more deeply in cells. Again, the ratio stayed stable during cell cycle progression. By contrast, a marked decrease of the YFP:CFP ratio, reflecting an open configuration, occurred when the protein was localized at the cleavage furrow. These differences were not observed with CFP–MELK K/R alone or with a mix of CFP–MELK K/R and YFP–MELK K/R, in spite of their accumulation at the cleavage furrow (Fig. 5C), thus excluding the occurrence of FRET due to auto-fluorescence or dimerization. These results demonstrate that xMELK conformational rearrangements occur during cell division and are correlated with its subcellular localization.

Overexpression of xMELK impairs accumulation of activated Rho GTPase at the division furrow

Indirect immunofluorescence and live imaging demonstrated that xMELK marks the division site shortly before furrow ingression. This regulation of xMELK localization was reminiscent to that described for the activated small GTPase Rho (Bement et al., 2005; Miller and Bement, 2009). Indeed, using a fluorescent probe allowing specific detection of activated Rho [GFP–rGBD (Benink and Bement, 2005)], it has been shown, in echinoderms and Xenopus embryos, that active Rho GTPase accumulates as a narrow band at the equatorial cell cortex presaging the division furrow before it ingresses (Bement et al., 2005). The similarity between the spatio-temporal localization of active Rho and xMELK prompted us to examine the effect of elevating xMELK levels on
activated Rho localization. As described previously, the GFP–rGBD fluorescent probe localized at the presumptive cleavage site before furrow ingression (Fig. 6). When coexpressed with xMELK K/R, which does not induce defective cell division, active Rho accumulated at the division site (Fig. 6). By contrast, when coexpressed with active xMELK, the GFP–rGBD band was not detected at the furrow (Fig. 6) and blastomeres still initiated furrow ingression that ultimately regressed. The fact that cells still present signs of furrow ingression suggests that either a low amount of cortically localized Rho is sufficient to allow the initiation of membrane ingression or that an alternative pathway, independent of Rho, allows initiation of cytokinesis. This issue is discussed further below. These data show that overexpression of xMELK perturbs accumulation of activated Rho at the cleavage furrow and provide a direct explanation for the cytokinesis defect induced by overexpression of xMELK.

Spatio-temporal xMELK conformational changes. (A) Intramolecular FRET for YFP–XMELK K/R-CFP. Fluorescent proteins are fused to the same xMELK K/R molecule allowing FRET from the donor CFP to the acceptor YFP. FRET depends on distance and orientation and thus indicates conformational changes. CFP is excited by 433 nm light, and then, after energy transfer, light emitted at 527 nm by YFP is detected. (B,C) Embryos were injected with YFP–XMELK K/R-CFP (B), xMELK K/R-CFP or a mix of xMELK K/R-CFP plus YFP–XMELK K/R (C). Surface views for CFP and colour-coded images of the YFP:CFP emission ratio (‘YFP/CFP’) are shown. White arrowheads indicate cleavage furrows. A confocal section at a distance of 18 μm relative to the blastomere surface is shown for time 18 (t18). Mean values of YFP:CFP emission ratios measured for three dividing cells at several subcellular locations are plotted versus time; error bars represent the s.d. The longitudinal brackets indicate the period of cytokinesis. Time is indicated in minutes. Scale bars: 50 μm.

xMELK copurifies with anillin, an essential cytokinesis component

xMELK and active Rho accumulate in a band that marks the presumptive division site shortly before furrow ingression. To analyse further this discrete cellular localization, we examined the localization of anillin, a key protein involved in cytokinesis, which has been shown to be localized at the equatorial cortex during anaphase in Caenorhabditis elegans (Maddox et al., 2005), Drosophila (Field and Alberts, 1995), Xenopus cultured cells (Straight et al., 2005) and embryos (Miller and Bement, 2009) as well as human cultured cells (Piekny and Glotzer, 2008). First, we tested by immunoprecipitation
whether the two endogenous proteins could be copurified. As expected, endogenous xMELK and anillin were immunoprecipitated, respectively, by affinity-purified antibodies against MELK and against Xenopus anillin and were not detectable when pre-immune serum was used (Fig. 7A). We found that endogenous xMELK was specifically co-immunoprecipitated with anillin and, reciprocally, anillin was co-immunoprecipitated with xMELK. Anillin and xMELK were not detected in immunoprecipitations made with pre-immune serum, and, in addition, β-tubulin was absent from all immunoprecipitates, demonstrating that xMELK and anillin co-immunoprecipitations are specific. Next, we examined the distribution of endogenous anillin in Xenopus early embryos with an affinity-purified antibody against Xenopus anillin (Straight et al., 2005). In the early blastula, anillin presented a distribution very similar to that observed for xMELK, active Rho, F-actin and MHC. Indeed, anillin was concentrated in a narrow band at the blastomere surface in addition to cortex (Fig. 7B). The band was already detectable at the equatorial cortex before furrow ingression started, demonstrating that anillin accumulates early at the cleavage site in Xenopus embryos. EGFP–anillin expressed in embryos confirmed immunofluorescence observations and showed that accumulation at the equatorial cortex is highly dynamic and precedes furrow ingression (data not shown). As the antibodies against both xMELK and anillin were raised in rabbits, the two endogenous proteins could not be examined simultaneously in the same cell. But coexpression of EGFP–anillin and xMELK K/R–CFP shows that the two proteins are colocalized at the equatorial cortex (Fig. 7C). Interestingly, in gastrula embryos, endogenous anillin (supplementary material Fig. S2B) and EGFP–anillin (supplementary material Fig. S4) were still detected as an equatorial band at the apical surface of dividing epithelial cells. This contrasted with the clear absence of xMELK, F-actin and MHC which, in the gastrula, were detected only at the cell cortex. Following EGFP–anillin localization in living embryos further showed that the basolateral membrane is already divided,
whereas the cell surface had only started to contract (supplementary material Fig. S4). Altogether, these results show that xMELK colocalizes and interacts with anillin, a crucial cytokinesis regulator, and reinforce the link between xMELK and cytokinesis.

Copurification of xMELK with anillin. (A) Endogenous xMELK and anillin co-immunoprecipitate. Protein extracts were prepared from cytokinetic one-cell embryos (input). Proteins were immunoprecipitated (IP) with antibodies against xMELK and anillin or pre-immune immunoglobulins (‘Pi’) and blotted with antibodies against anillin, xMELK or β-tubulin. (B) Endogenous anillin is localized at the equatorial and lateral cell cortex in Xenopus embryos. Blastula stage-7 albino embryos were fixed and processed for immunofluorescence with antibody to anillin. Single optical sections are shown from the cell surface (Z0, left) towards deeper planes (the distance relative to Z0 is indicated in μm). The cell surrounded by a dotted line is further magnified and shown in the row below. Scale bars: 100 μm. (C) Colocalization of xMELK K/R-CFP and EGFP–anillin at the division furrow. Pictures were merged to visualize colocalization of xMELK K/R-CFP and EGFP–anillin at the division furrow (merge). Scale bar: 100 μm. (D) Schematic representation of the transition in the mode of cytokinesis during early development. Cubes represent cytokinetic embryonic cells. In large cells of blastula embryos, xMELK, F-actin, active Rho (‘act. Rho’), MHC (all in red) and anillin (green) are localised at the cell cortex and at the cleavage furrow. The furrow is an arc that expands in an apical-to-basal direction (arrows). In contrast, in smaller cells of gastrula, only anillin forms a ring at the equatorial cortex corresponding to the furrow which ingresses in a basal to apical direction. At this developmental stage, xMELK is still localized at the cortex.

Discussion

In this study, we investigated the function of the xMELK protein kinase in cleaving Xenopus embryos. We show that xMELK marks the division site, copurifies with the cytokinesis factor anillin and demonstrate that alterations in its levels of expression affect the progression of cytokinesis through mislocalization of RhoA.

By knockdown of xMELK before fertilization, we show that xMELK is the product of a maternal-effect gene necessary for completion of cytokinesis. Surprisingly, overexpression of xMELK also results in abortive cytokinesis. Numerous studies have documented the unstable properties of cytokinesis in animal cells. The reversibility of cell division highlights the requirement to maintain constriction until the daughter cells are topologically distinct. The actomyosin-based contractile ring produces the force necessary for ingression of the plasma membrane. It is established that cytokinesis requires polymerization of actin filaments (F-actin) as well as depolymerization. This requirement for F-actin dynamics is illustrated by the deleterious effects of actin-depolymerizing drugs (Merriam et al., 1983) or inactivation of cofilin, an F-actin-depolymerising and -severing factor (Abe et al., 1996). Accordingly, deregulation of signalling pathways that operate on the F-actin polymerization–depolymerization equilibrium eventually affects cytokinesis. As demonstrated in Xenopus embryos, both increasing and decreasing the activity of RhoA, leading, respectively, to increasing and decreasing the amount of F-actin, inhibit cytokinesis (Drechsel et al., 1997). The fact that xMELK knockdown impairs furrow ingression indicates that xMELK is important for cytokinesis after its initiation. The overexpression of xMELK is also deleterious for cell division. This suggests that a misregulated phosphorylation of a putative xMELK substrate involved in cytokinesis could block cytokinesis progression and that xMELK must be inactivated for cytokinesis to proceed. The finding that perturbing the levels of xMELK leads to a default in furrow ingression rather than a failure of cytokinesis initiation suggests that xMELK participates in the maintenance of the cytokinetic ring constriction. Moreover, the fact that overexpression of xMELK leads to a default in accumulation at the division site of active RhoA, which is crucial for the function of the acto-myosin ring, could explain the detrimental effect of overexpression of xMELK on the constriction of the cytokinetic ring.

Interestingly, we noticed that, in spite of a large decrease in the accumulation of active Rho at the division site induced by overexpression of wild-type xMELK, blastomeres still initiated cytokinesis. We cannot rule out the possibility that a low level of active Rho localized at the equatorial cortex is sufficient to initiate cell division; however, this would be insufficient to maintain constriction. By contrast, this observation suggests that, in Xenopus embryos, an alternative Rho-independent pathway might contribute to early steps of cytokinesis. In LLC-Pk1 tissue-culture cells that adhere tightly to the substratum, it was observed that the Clostridium botulinum C3 transferase, which ADP-ribosylates and thereby inhibits Rho, does not prevent cytokinesis in the majority of treated cells (Murthy and Wadsworth, 2008). Although surprising, this result was in agreement with a similar observation reported previously (O'Connell et al., 1999). Moreover, Yoshizaki and colleagues (Yoshizaki et al., 2004) have shown by using mammalian cells (HeLa and Rat1A) that activation of RhoA is tissue specific, leading to differential effects of C3 transferase treatment on cytokinesis. In Dictyostelium discoideum myosin-II-null cells, cytokinesis can still proceed by furrow formation, which relies on traction forces when cells are anchored on the substratum (Neujahr et al., 1997; Zang et al., 1997). It was also reported that certain adherent mammalian cells can divide by a contractile-ring-independent, adhesion-dependent process when the contractile ring function is compromised (Kanada et al., 2005). We hypothesize that, in adherent cells of the Xenopus embryo, a similar mechanism could operate to allow the formation of an abortive furrow when accumulation of active Rho at the division site is compromised by overexpression of xMELK.

In cleaving embryos, xMELK shares remarkable properties with active RhoA, actin, myosin heavy chain and anillin. One of the most noteworthy resemblances between these proteins is their accumulation at the presumptive division site before the furrow starts to ingress. Indeed, like the discrete RhoA activity zone that prefigures the division site (Bement et al., 2005), we have shown that xMELK and anillin dynamically accumulate in a narrow band at the equatorial cell cortex shortly before furrow ingression. Later in development, in gastrula embryos, cells still accumulate anillin at the equatorial cortex in contrast to xMELK, actin and MHC, which are not concentrated at this site, although they accumulate at the cell cortex. Thus, the equatorial band is not specific for active RhoA and appears as a specialized structure that concentrates several cytokinesis molecules in rapidly dividing large cells. We have precisely determined that xMELK is no more concentrated at the equatorial cortex between stage 7 and stage 9. Therefore, the decrease in xMELK concentration at the equatorial cortex correlates with the MBT, during which important modifications occur, such as cell cycle remodelling and the commencement of zygotic gene expression. Interestingly, bundles of microtubules that form a structure, termed the furrow microtubule array [FMA (Danilchik et al., 1998)], at the base of the cleavage furrow that are probably involved in furrow progression were observed until stage 7. Together, these data indicate that profound modifications in the mode of cytokinesis occur at the MBT. The change in xMELK accumulation at the equatorial cortex might depend on a factor and/or a posttranslational modification regulated at the MBT. The high xMELK concentration at the equatorial cortex appears to be specific to large cells. Indeed, during the cleavage period, the size of embryonic cells decreases by about two orders of magnitude, ranging from more than a millimetre in one-cell embryos to ~20 μm in gastrula cells. The preferential accumulation of xMELK, F-actin, MHC, anillin and active RhoA at the equatorial cortex in blastomeres might reflect the higher forces necessary for membrane ingression in large compared with small embryonic cells, as proposed by Wang (Wang, 2005). Interestingly, in C. elegans and sea urchin embryos, it was shown that the duration of cytokinesis is independent of the original cell size, with large cells dividing in the same amount of time as exhibited by small cells (Carvalho et al., 2009; Mabuchi, 1994). Thus, the mechanism of cytokinesis scales with cell size, leading to a model of structural memory of the contractile ring (Carvalho et al., 2009). Nevertheless, the molecular base of the structural memory is unknown. However, it was proposed that the cytokinesis ring comprises contractile units. The number of these units, the base of which would be F-actin, would be proportional to the original cell size and they would shorten during constriction of the ring. Contractile units could explain the structural memory and the proportionality between the constriction rate and the initial cell size observed during cleavage of embryonic cells (Carvalho et al., 2009). In large cells, some molecules might be specifically recruited at the cell division furrow, which could contribute to potentiate the furrow constriction rate. As xMELK is accumulated preferentially at the equatorial cortex specifically during cleavage and as modification of its levels alters the progression of cytokinesis, it might be specifically involved in sustaining cytokinesis in large embryonic cells.

We also established in this current study that an inversion in furrow ingression occurs between late-blastula and gastrula stages (Fig. 7D). In gastrula cells, the division furrow progresses from the basolateral towards the apical membrane, whereas, in the blastula, it is the opposite. This in vivo observation is in agreement with the asymmetric ingression already described in mammalian epithelial cells cultured in vitro (Reinsch and Karsenti, 1994). In C. elegans, it was shown that asymmetric ingression of the division furrow during the first embryonic division depends on several factors, including anillin, septin and the RNA-binding protein CAR-1 (Audhya et al., 2005; Maddox et al., 2007). Our study shows that asymmetric furrow ingression also occurs in a vertebrate organism and therefore suggests that it is potentially important. The reason for the inversion in ingression polarity during development is unknown, and it will be of great interest to test whether this feature was conserved through evolution.

Anillin is a key protein involved in cytokinesis that stabilizes the division furrow (Hickson and O'Farrell, 2008a). Our finding that, in Xenopus embryos, anillin accumulates at the equatorial cortex before the furrow starts to ingress is in agreement with a previous report showing that anillin plays a role early in cytokinesis (Hickson and O'Farrell, 2008b). Interestingly, similarly to active RhoA and xMELK, accumulation of EGFP–anillin at the equatorial cortex is very dynamic, appearing immediately as a band of defined width and propagating along the equatorial cortex. This contrasts with EGFP–anillin in Drosophila S2 cells, where two steps in its cortical distribution have been identified: first, anillin localizes to the cell cortex at metaphase and is focused to the equator; and, second, anillin disappears from the poles during anaphase. This raised the possibility that the mechanism leading to anillin concentration at the equatorial cortex in insect cells is not conserved in Xenopus or, more likely, that the equatorial band of Xenopus early embryos is not strictly the equivalent of the equatorial band reported in Drosophila. Contrary to F-actin, MHC and xMELK, anillin is still localized at the equatorial cortex in post-cleavage embryos, suggesting a sustained requirement for a high concentration of anillin at the division site for cytokinesis, whereas other cytokinesis molecules are no more preferentially accumulated at this site. Studies utilizing diverse organisms have reported that anillin interacts with several molecules involved in cytokinesis, including actin (Field and Alberts, 1995), non-muscular myosin II MHC (Straight et al., 2005), RhoA (Piekny and Glotzer, 2008) and MgcRacGAP (D'avino et al., 2008; Gregory et al., 2008). Interestingly, xMELK and anillin co-immunoprecipitate, demonstrating that the two proteins interact either directly or indirectly. Altogether, our results indicate that, during cleavage of the large cells of early embryos, anillin probably contributes to the concentration and/or stabilization of cytokinesis factors, including xMELK, at the equatorial cortex.

In conclusion, our study demonstrates that important modifications in the cytokinesis mechanism occur during the early development of a vertebrate organism (Fig. 7D). The specialization of the cytokinetic process has been reported in distinct differentiated cell types of post-cleaving Xenopus embryos (Kierserman et al., 2008). Here, we show that important modifications occur in early-cleaving embryos. Localization at the equatorial cortex, copurification with the cytokinesis factor anillin and the deleterious effect on the progression of cytokinesis of altered xMELK levels all indicate that xMELK participates in cytokinesis in large embryonic cells.

Materials and Methods

Preparation of Xenopus embryos

xMELK knockdown

Knockdown before fertilization was performed as described by Heasman and colleagues (Heasman et al., 1991). Briefly, full-grown oocytes of pigmented frogs were manually dissected in oocytes culture medium [OCM; Liebovitz L-15 medium (Sigma) diluted 1:2 with sterile deionized water, 0.32 g/l bovine serum albumin (Sigma), 5% penicillin–streptomycin (Sigma)]. Oocytes were microinjected with 2 ng/oocyte (volume of injections was 13.8 nl) of HPLC-purified antisense oligos (Eurogentec) AS9: GTCTCTGCTCTACAAAGAG, AS11: CGCTCTTTCTCGATCCGGAC and AS Co: CAAGTTATAGTATTCTGATT (underlined are phosphorothioate-modified bases) and cultured at 16°C. The day after injection, oocytes were placed at 21°C and maturation was induced by treatment with 1 μM progesterone (Sigma) overnight. Matured oocytes were stained with vital dyes to distinguish the different set of oocytes and transferred into the same host laying female. Finally, laid eggs were in vitro fertilized and processed for subsequent analysis.

Construction of plasmids

pT7T-xMELK(Eg3) was constructed previously (Blot et al., 2002). pT7T-xMELK K/R was obtained by PCR from the pET21a-Eg3 K/R plasmid (Blot et al., 2002) with oligos xMELK-20 and xMELK-21 (all primers are listed in supplementary material Table S1). PCR product was cut with BglII and XbaI restriction enzymes and ligated into pT7T cut with BglII and SpeI. pT7T-NEGFP-xMELK allowing expression of EGFP–XMELK was constructed by PCR using the pEGFPN1-XlEg3 (Chartrain et al., 2006) plasmid with the primers xMELK-71 and pEGFP-C1-3′. The PCR product was digested with NruI and XbaI and ligated into pT7T vector (Krieg and Melton, 1984) at EcoRV and SpeI sites. To construct the pT7T-NEGFP-xMELK K/R plasmid, a DNA fragment containing the K/R mutation was excised from the pT7T-xMELK K/R plasmid and was exchanged in the pT7T-NEGFP-xMELK plasmid. To construct the pT7T-NEGFP-Xlanillin, the Xenopus anillin open reading frame was PCR amplified from pAFS217 (Straight et al., 2005) using anillin-1 and anillin-2 primers. The PCR product was cut with SpeI and ligated in pT7T digested with EcoRV and SpeI to generate pT7T-Xlanillin. EGFP ORF amplified from pEGFPN1 using GFP-5 and GFP-7 primers and cut with BglII and EcoRV and was ligated into pT7T-Xlanillin cut with BglII and EcoRV. To obtain the plasmid allowing expression of the FRET-based probe, pT7T-YFP-xMELK K/R-CFP, YFP was PCR-amplified from pEYFP vector (Clontech) with EYFP-1 and EYFP-2 primers, xMELK K/R ORF was PCR-amplified from pET21a-XlEg3 K/R plasmid with xMELK-72 and xMELK-75 primers and CFP was PCR-amplified from pECFP vector (Clontech) with ECFP-3 and ECFP-2 primers. Note that primers EYFP-2 and ECFP-3 each introduce five consecutive glycine residues, allowing free orientation of fluorescent proteins. The PCR products were cut, respectively, with BglII, BglII and XhoI and XhoI and SpeI. Fragments were ligated into pT7T open with BglII and SpeI restriction enzymes. The pT7T-xMELK K/R-CFP was constructed following the same strategy except that the YFP fragment was omitted in the ligation. The pT7T-YFP-xMELK K/R plasmid was obtained following the same strategy with xMELK K/R ORF amplified with xMELK-72 and xMELK-21 primers. All constructs were subsequently verified by sequencing.

Microinjection of in vitro transcribed mRNAs

In vitro transcriptions were performed with mMessage mMachine transcription kits following the manufacturer's instructions (Ambion). mRNAs were microinjected in one blastomere of two-cell stage embryos (volume of injections was 13.8 nl) and placed at 16°C until observation.

Imaging

Only embryos obtained from albino females were used for live imaging. Imaging was performed on a Leica SP2 confocal microscope using a ×20 HC PL APO-ON 0.7, ×40 HC Plan-APO-ON 1.25 and a ×63 HCX Plan-APO-ON 1.4 oil-immersion objective lens (Microscopy platform, IFR140). Images and movies were obtained by projections of optical sections using the ImageJ software (Rasband, W. S., http://rsb.info.nih.gov/ij/). Figures were assembled in Adobe Photoshop and Adobe Illustrator (Adobe Systems).

Second polar body extrusion

To prepare full-grown oocytes (stage VI), fragments of Xenopus ovaries were incubated in OR2 medium (10 mM Hepes, pH 7.6, 82.5 mM NaCl, 2.5 mM KCl, 1 mM CaCl2, 1 mM MgCl2) containing 0.4 mg/ml of Dispase II (Boehringer) for 3 hours at 21°C. Next, ovary fragments were incubated in OR2 containing 100 units/ml of collagenase (Sigma) in the absence of Ca2+ for 1 hour. Recovered oocytes were then washed extensively in OR2. Full-grown oocytes (stage VI) were microinjected, as described above. In vitro maturation was triggered by 15 μM of progesterone (Sigma). Matured oocytes were then prick activated and fixed in methanol 30 minutes later. To stain DNA, DAPI was added at a final concentration of 10 μg/ml for 1 hour. After washing in PBS, oocytes were mounted in Vectashield (Vector) for observations. Imaging was performed on a DMRXA Leica microscope coupled to a Q550 CW image-analysis system (Leica) at the Microscopy platform, IFR140.

Acknowledgments

We thank A. F. Straight for anillin cDNA, purified anillin, anti-anillin and anti-myosin heavy chain antibodies; R. Stick for anti-lamin antibody; J. Ellenberg for GFP–ABD and W. M. Bement for GFP–rGBD. We are grateful to M. Tramier for helpful discussions on FRET, to Y. Audic and C. Wylie for their help with the transfer method and to J. Kubiak, H. McNeill and Ankush Garg for critical reading of the manuscript. This work was supported by the Ligue Départementale contre le Cancer (22 et 35), the ARC and INCa.

(1997). Myosin II-independent processes in mitotic cells of Dictyostelium discoideum: redistribution of the nuclei, re-arrangement of the actin system and formation of the cleavage furrow. J. Cell Sci.110, 123-137.

Marian Blanca Ramírez from the CSIC in Spain has been studying the effects of LRRK2, a protein associated with Parkinson’s disease, on cell motility. A Travelling Fellowship from Journal of Cell Science allowed her to spend time in Prof Maddy Parson’s lab at King’s College London, learning new cell migration assays and analysing fibroblasts cultured from individuals with Parkinson’s. Read more on her story here.

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